PRM - Unstable signals

PRM - Unstable signals melanieH  2020-06-15

Dear all,

I would like to have your advices regarding my issue.
We are working with a newly acquired Q-exactive HF-X and I am trying to setup a PRM method to quantify one bacterial protein (matrix : bacterial culture supernatants).
My issue is the signal that I obtain in scheduled PRM method, it seems really unstable but I am not sure if it comes from my method (actually pretty basic, following Thermo recommendations) which could be too much for just a low number of peptides [PRM R=30000, AGC t = 1e5, IT max = 60 ms, isolation window = 1.6m/z ) or if it could come from the instrument itself.
I attached a pdf file with some screenshots. I have more data but the results are similar for all the runs operated in scheduled mode, with or without FullScan. As you can see the signals seem "smoother" when the method is unscheduled.
I am asking if it could come from the instrument because we had once a problem with a lens in the quad that fell down during a cleaning session by the Thermo technician...

All advices are welcome.


Nick Shulman responded:  2020-06-15
Can you send us your Skyline document and a couple of your raw files (one smooth, and one jagged)?

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

You can upload that .zip file and your .raw files here:

-- Nick
melanieH responded:  2020-06-15
Thanks for your reply,

I followed your instructions and attached the zip file to this message.

Nick Shulman responded:  2020-06-16

Can you send us the files "9754(940)uPRM_20200612104946.raw" and "9754(940)fMStPRM1.raw"?

You can upload those files here:

Probably when we take a look at the spectra inside those files, we will be able to tell you why the first file has smooth chromatograms and the second one has jagged chromatograms.
-- Nick
melanieH responded:  2020-06-16

I uploaded a zip file (20200617_9754(940)_3) containing 3 raw files : 1 unscheduled method ("uPRM"), 1 scheduled method ("tPRM") and 1 scheduled method with a Full MS scan ("fMStPRM").

Thanks for your help and time,

Nick Shulman responded:  2020-06-17
Thanks for sending those raw files.
Unfortunately, I do not know what is going wrong.

One thing that I did notice is that in the unschedules raw file, the scan header "Last locking (sec)" is always a number very close to zero, whereas in the other .raw files, it is an ever increasing number in the thousands.

I do not know very much about how to use a mass spectrometer. The usual things that might be going wrong, such as the observed m/z's being close to the edge of the extraction window, are not happening. All of the peaks in the spectra are properly centered at the correct m/z's.

This looks like it might be the thing people call "spray instability". Have you tried collecting data again using the unscheduled method again, to see whether the reason the unscheduled method looked good before was because it was collected before something bad happened to your column?

Maybe someone else on this board with more practical mass spectrometer experience could help.
-- Nick